Multiple post-translational modifications contribute to regulate the activity of many essential cellular factors. These modifications yield a quick cellular response to change the activity of the target proteins by modifying among others, their capacity to associate with multiple partners, connect with downstream factors, alter their sub-cellular localisation or promote variations in protein synthesis and degradation. Amongst the different strategies to control protein activity, probably the most drastic one is protein degradation, because it inactivates all protein functions. In eukaryotic cells, the ubiquitin-proteasome system (UPS) drives one of the most important proteolytic activities.
As its name implies, ubiquitin is a ubiquitous and highly conserved protein found in all eukaryotic tissues. The ubiquitin molecule can be found free or conjugated to protein substrates, where it modifies the biochemical properties of the target protein drastically. The attachment of ubiquitin to a protein-substrate is achieved by a cascade of thiol-ester reactions, mediated by an ubiquitin activating enzyme (E1), an ubiquitin conjugating enzyme (E2) and an ubiquitin ligase (E3) as it is shown schematically in FIG. 1.
Ubiquitylation (or ubiquitination) is defined as the process whereby a lysine residue in a substrate molecule is covalently bonded to ubiquitin. Modification of the substrate may be in the form of mono-ubiquitylation (possibly at multiple sites—this is referred to as multiple monoubiquitylation) or as poly-ubiquitylation. Depending on the number of ubiquitin molecules attached to the protein substrate and the lysine residues on the ubiquitin moieties involved in the formation of ubiquitin chains, the destiny of a protein will be different.
To recognise various forms of ubiquitylated targets a limited number of protein effectors will specifically interact with such modified proteins working as “ubiquitin receptors”. Such receptor/effector proteins will directly mediate or connect with a function depending on the particular modification and/or modified protein. The property of ubiquitin binding is most often localized to a modular domain, which independently can recognize and interact with ubiquitin.
Ubiquitin binding domains (UBDs) are a diverse family of structurally related dissimilar protein modules which bind mono- and poly-ubiquitin. The first protein motif characterized to bind ubiquitin non-covalently was found in the proteasomal subunit S5a (rpn10 in yeast). S5a is a part of the 19S proteasome regulatory particle, where it links the base to the lid. The ubiquitin binding property of S5a pertains to two independent Ubiquitin Interacting Motifs (UIMs), which are short α helical regions. Following the discovery of the UIM, many other ubiquitin binding domains (UBDs) were characterized, and today at least 16 different motifs have been described, among them the UBA (Ubiquitin Associated domain), UIM (Ubiquitin Interacting Motif), MIU (Motif Interacting with Ubiquitin) domain, DUIM (double-sided ubiquitin-interacting motif), CUE (coupling of ubiquitin conjugation to ER degradation) domain, NZF (Np14 zinc finger), A20 ZnF (zinc finger), UBP ZnF (ubiquitin-specific processing protease zinc finger), UBZ (ubiquitin-binding zinc finger), UEV (ubiquitin-conjugating enzyme E2 variant), PFU (PLAA family ubiquitin binding), GLUE (GRAM-like ubiquitin binding in EAP45), GAT (Golgi-localized, Gamma-ear-containing, Arf (ADP-ribosylation-factor) binding), Jab/MPN (Jun kinase activation domain binding/Mpr1p and Pad1p N-termini), UBM (Ubiquitin binding motif) and a Ubc (ubiquitin-conjugating enzyme). Most UBDs are 20-40 amino acids long structural motifs without sequence conservation that are found in all eukaryotes. Amongst these motifs, the UIM and the UBA domain are the two best characterized. An important question concerning the trafficking and presentation of modified proteins to the proteasome is how these processes are regulated. Understanding how modified substrates are brought to the proteasome for subsequent degradation may result in the identification of new points of possible therapeutic intervention.
One of the best described UBDs is the UBA domain. The UBA domain family exhibits poor sequence homology, but are structurally well conserved as compact three helix bundles. UBA domains are classified into four different groups, depending on their ubiquitin binding properties. Class 1 and 2 are defined as binding K48 and K63 poly-ubiquitin chains, respectively, class 3 does not bind poly-ubiquitin chains, and class 4 does not exhibit any particular specificity for chain linkage in poly-ubiquitin, whilst binding equally strongly to monoubiquitin. The physiological purpose of the UBA domain will necessarily depend on the nature of the protein which it is found in—however, several UBA domain containing proteins have been suggested to serve as factors shuttling ubiquitylated substrates to the proteasome (e.g. hHR23A, p62, Dsk2). This particular function would be achieved by binding to ubiquitylated substrates through the UBA domain, and binding to the proteasome via another domain, usually an Ubiquitin Like domain (UBL). These domains share homology with ubiquitin, and many have been shown to interact with the proteasome through S5a. In addition to S5a, all other helical UBDs have also been reported to interact with ubiquitin through its hydrophobic surface patch. Hhr23A and Hhr23B (human homologs of yeast proteins Rad23A/B) are examples of UBA/UBL proteins, containing an N-terminal UBL domain and two UBA domains. The function of Hhr23A may be regulated by competition/cooperation arising from intramolecular or intermolecular interactions between its UBAs and UBL. Intramolecular UBL-UBA binding has been suggested to result in a closed domain organization, which may be opened up by binding of S5a UIMs to the Hhr23A UBL, disrupting Hhr23A intramolecular contacts.
There are several documents in the state of the art which describe methods for the isolation and purification of ubiquitylated proteins. For example, document WO04106514 describes a method of recovering protein having been ubiquitylated, comprising recovering ubiquitylated protein with the use of a specific antibody capable of recognizing ubiquitin chains through immunological means, such as affinity chromatography or immunoprecipitation. However, ubiquitin antibodies are expensive and do not show the protective effect reported for some UBA domains which may contribute to increase the efficiency of purification (Gwizdek C, et al. Proc. Natl. Acad. Sci USA., 2006, 103:16376-81). Cavey et al. (Cavey, Jr. et al. www.biochemistry.org/meetings/abstracts/BS2006/BS20060356) have described a method for the purification of ubiquitylated proteins using the UBA domain from p62 protein followed by elution of ubiquitylated proteins. Such method presents a low affinity-purification of ubiquitylated proteins since a single UBA domain is employed for such purification. Mayor and Deshaies (Methods Enzymol. 2005; 399:385-92) have described an affinity purification protocol using a budding yeast strain expressing hexahistidine-tagged ubiquitin. Such method is a two-step purification method that uses a cell expressing hexahistidine-tagged ubiquitin, thus needing a first modification of the ubiquitin protein and moreover, using of a two-step purification protocol. Peng J. and Cheng D. (Methods Enzymol. 2005; 399: 367-81) have also described a purification protocol for the purification of ubiquitylated proteins in which the ubiquitin protein is modified by adding a His-tag at its N-terminal end thus, needing a first modification of the ubiquitin protein. Layfield et al (Proteomics. 2001, 1:773-7) have described an immobilised glutathione-S-transferase (GST)-S5a fusion protein to purify poly-ubiquitylated proteins from mammalian tissues. Such a proteasomal fusion protein may be mostly suitable for purification of proteins containing Lys48-linked ubiquitin chains. WO03/049602 describes methods for establishing a protein expression profile of a biological sample. Such method comprises purification of ubiquitylated proteins by means of using the UBA domains of an ubiquitin binding protein, such as Rad23, said UBA domains being assembled as multimeric forms of UBA affinity matrices. Thus, said method only describes the use of UBA domains in said multimeric proteins, and moreover, said multiple domains as described therein include no more than two of such UBA domains. On the other hand, BIOMOL commercialises a kit for the isolation and enrichment of ubiquitylated proteins through use of a high-binding affinity matrix. Said matrix comprises single UBDs (ubiquitin binding entities) for binding to ubiquitylated proteins.
Thus, there is a need for the development of a new reliable method for the isolation and purification of ubiquitylated proteins with high affinity for such proteins and which would facilitate further analysis of said isolated protein. Such tools should allow capturing said modified proteins in vitro and/or in living cells.